Antibiotic resistance surveillance becomes more and more important in a global situation of increased occurrence and spread of resistance genes among bacterial pathogens. National as well as international surveillance of antibiotic resistance is thus urgently needed in the present increase of resistance worldwide.
Antimicrobial susceptibility testing (AST) constitutes one of the most important methods of analysis in a clinical microbiology laboratory. Correct information regarding the most appropriate antimicrobial agent(s) to be used as empirical therapy in for example an acute clinical situation of an infected patient requires thorough knowledge of the antimicrobial susceptibility levels in relation to the bacterial species and the type of infection in question.
In addition, when a causative bacterial strain has been isolated from the site of infection the antimicrobial test results provide a rational basis for either continued therapy or a change to a more effective drug.
Different types of susceptibility tests can be used to test the antimicrobic susceptibility of a microorganism. One type of susceptibility test is the disk diffusion test. This is a standardized test, in which a plate containing a growth medium in agar gel is inoculated with a microbial isolate and one or more disks impregnated with fixed concentrations of antibiotics are placed thereon. After appropriate incubation, the diameter of zones of inhibition around the disks (if present) are registered in order to determine the sensitivity of the inoculated microorganism to the particular antimicrobial agent impregnated in each disk.
Another type of susceptibility test is the broth microdilution test. In this type of test, dilutions of antibiotics are prepared in tubes or microwells. Each tube or well with various concentrations of antibiotics, usually as a twofold dilution series, is inoculated with a standardized suspension of a particular microorganism. After incubation, the wells or tubes are examined for turbidity, haze and/or pellet and compared with a growth control as well as a non-inoculated control. The minimum concentration of antimicrobial agent that prevents visible microbial growth is calculated as the Minimal Inhibitory Concentration (MIC).
In spite of the availability for several years of automated microdilution methods, the disk diffusion method is still the most widely used susceptibility testing procedure in most countries. The test is performed according to some standardized methodology issued by a reference group, such as NCCLS (National Committee for Clinical Laboratory Standards), SRGA (Swedish Reference Group for Antibiotics), the French “Comité de l'Antibiogramme de la Société Francaise de Microbiologie”, the British “Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy”, the Australian “ASIG Antimicrobials Special Interest Group”, the German DIN group, etc.
The results of this test are semi-quantitative. In. 1979 the SRGA introduced the 3 susceptibility categories which are still in use today: S (susceptible), I (intermediate) and R (resistant) in dependence of the size of the inhibition zone.
However, the definitions and interpretive criteria for resistance vary with the guidelines adhered to as do those test results which are influenced by differences in test performance. The differences are particularly paramount for disk test results, which means that the majority of antibiotic susceptibility test results cannot be utilized for surveillance purposes.
Most surveillance efforts have relied on MIC tests because of the higher accuracy of such methods. The adherence to disk diffusion standards are often incomplete and the results unreliable. In one investigation using the disk method, all participating laboratories sent their bacterial isolates to one single laboratory where the disk tests were performed in order to ensure comparable results from the different geographical regions. Such an approach will be impossible if resistance surveillance is to be performed on a larger scale globally.
In general, organisms are considered susceptible to an antibiotic when they are inhibited by concentrations easily achieved in vivo and when the clinical efficacy has been documented. This implicates a correlation between the MIC and the clinical outcome. Similarly, organisms are termed resistant when concentrations required for inhibition are higher than those easily obtained in vivo, indicating that treatment with this antibiotic is not likely to be successful.
The statistical parameters for the inhibition zones obtained for individual bacterial species have been investigated (Kronvall et al., APMIS 99:887-892, 1991). At this time the bacterial species studied did not exhibit a considerable resistance to the antibiotic of choice, gentamicin. In order to be able to study a homogenous population any resistant outliers were eliminated, and the statistical parameters were only determined for those strains which belonged to the susceptible population.
When antibiotic susceptibility test results, such as inhibition zone diameter values, are analyzed separately for each antibiotic and bacterial species, including resistant strains, some special features become apparent. The position of a normal wild-type population of zone diameters in a given laboratory will stay remarkably stable over the years, reflecting the stability of a routine method in daily use. However, when results are compared between different laboratories, the position of a normal population might vary considerably. These inter-laboratory differences are the main reason for the relative lack of accuracy of the disk diffusion test.
One approach to solve the problem of interlaboratory variation has been to introduce a reference or control strain, which serves as a calibrator strain. Control strains are recommended by reference authorities for quality control and therefore such strains are usually already available in the laboratory.
A calibration is a regular procedure in any clinical chemistry test and involves the use of a calibrator with a known concentration of the substance to be measured. The calibration of the test in the individual laboratory using their own equipment and reagents is then controlled using control samples, either internal controls or from an external quality control agency.
One method for calibration of the disk diffusion test is called “reference strain corrected breakpoints” (or “control strain peak correction”) and requires the regular testing of an international reference strain in the laboratory (Kronvall et al., Antimicrob. Agents Chemother. 32:1484, 1988). The position of the zone diameter peak in the reference laboratory has to be known and the difference in mm value from this peak value to the interpretive break-points can be calculated. A similar relation should exist in the individual laboratory between its control peak value and the breakpoints, which should be adjusted accordingly. This method improved the accuracy of the disk test by reducing false-resistant interpretations from 4.4% to 2.3%.
A further improvement was obtained by using a second method, single strain regression analysis (Kronvall, J. Clin. Microbiol. 16:784, 1982). This method, called SRA, provides a calibration of the test, which is truly species-related and laboratory-specific. However, it both requires especially prepared disks with different disk potencies and computer programs for the calculations. This method further reduced false-resistant results down to 0.14% in the studies referred to above.